3′-epimeric k-252a derivatives

ABSTRACT

Compounds defined by the following general structure are disclosed:                    
     These compounds display pharmacological activities, including inhibition of tyrosine kinase activity and enhancement of the function and/or survival of trophic factor responsive cells, e.g., cholinergic neurons.

CROSS-RELATED REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.09/223,518, filed Dec. 30, 1998, now U.S. Pat. No. 6,093,713, whichclaimed benefit of provisional application Ser. No. 60/070,263, filedDec. 31, 1997.

FIELD OF THE INVENTION

The field of the invention is pharmaceutical chemistry.

BACKGROUND OF THE INVENTION

K-252a is an indolocarbazole whose stereochemistry is shown below(Formula I):

K-252a inhibits protein kinase C (PKC), which plays a role in regulatingcell functions. K-252a has various activities, e.g., inhibiting smoothmuscle contraction (Jap. J. Pharmacol. 43 (suppl.): 284, 1987),inhibiting serotonin secretion (Biochem. Biophys. Res. Commun. 144: 35,1987), inhibiting elongation of neuraxone (J. Neurosci. 8:715, 1988)inhibiting histamine release (Allergy 43:100, 1988), inhibiting smoothmuscle MLCK (J. Biol. Chem. 263:6215, 1988), anti-inflammatory action(Acta Physiol. Hung. 80:423, 1992), and promotion of cell survival (J.Neurochem. 64:1502, 1995). K-252a also inhibits IL-2 production (Exper.Cell Res. 193:175-182, 1991). The total synthesis of the natural (+)isomer of K252a and its enantiomeric (−) isomer (all three chiralcarbons of the sugar moiety inverted), has been achieved (Wood et al.,J. Am. Chem. Soc. 117:10413, 1995; and WO 97/07081).

SUMMARY OF THE INVENTION

We have discovered that certain 3′-epimeric derivatives of K-252a arebiologically active. These compounds have the following general formula(Formula II):

wherein:

R¹ and R² independently are:

hydrogen; lower alkyl; halogen; acyl; nitro;

sulfonic acid;

—CH═NR⁴, wherein R⁴ is guanidino, heterocyclic, or —NR⁵R^(6,) wherein R⁵or R⁶ is hydrogen or lower alkyl, and the other is hydrogen, loweralkyl, acyl, aryl, heterocyclic, carbamoyl or lower alkylaminocarbonyl;

—NR⁵R⁶;

—CH(SR⁷)₂ wherein R⁷ is lower alkyl or alkylene;

—(CH₂)_(j)R⁸, wherein j is 1-6, and R⁸ is halogen; substituted aryl;unsubstituted aryl; substituted heteroaryl; unsubstituted heteroaryl;N₃;

—CO₂R⁹, wherein R⁹ is hydrogen, substituted lower alkyl, unsubstitutedlower alkyl, substituted aryl, unsubstituted aryl, substitutedheteroaryl, or unsubstituted heteroaryl;

—C(═O)NR¹⁰R¹¹, wherein R¹⁰ and R¹¹ independently are hydrogen,substituted lower alkyl, unsubstituted lower alkyl, substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,substituted aralkyl, unsubstituted aralkyl, lower alkylaminocarbonyl, orlower alkoxycarbonyl, or R¹⁰ and R¹¹ are combined with a nitrogen atomto form a heterocyclic group;

—OR¹², wherein R¹² is hydrogen, substituted lower alkyl, unsubstitutedlower alkyl, substituted aryl, unsubstituted aryl; or —C(═O) R¹³,wherein R¹³ is hydrogen, NR¹⁰R¹¹, substituted lower alkyl, unsubstitutedlower alkyl, substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl, substituted aralkyl, orunsubstituted aralkyl;

—NR¹⁰R¹¹;

—C(═O)R¹⁴, wherein R¹⁴ is hydrogen, lower alkyl, substituted aryl,unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl;

—S(═O)_(r)R¹⁵, wherein r is 0 to 2, and R¹⁵ is hydrogen, substitutedlower alkyl, unsubstituted lower alkyl, substituted aryl, unsubstitutedaryl, substituted heteroaryl, unsubstituted heteroaryl, substitutedaralkyl, unsubstituted aralkyl, thiazolinyl, (CH₂)_(a)CO₂R¹⁶, wherein ais 1 or 2, and R¹⁶ is hydrogen or lower alkyl, or —(CH₂)_(a)C(═O)NR¹⁰R¹¹;

—OR¹⁷, wherein R¹⁷ is hydrogen, lower alkyl, or —C(═O)R¹⁸, wherein R¹⁸is substituted lower aIkyl, unsubstituted lower alkyl, substituted aryl,or unsubstituted aryl;

—C(═O)(CH₂)_(j)R¹⁹, wherein R¹⁹ is hydrogen, halogen, NR¹⁰R¹¹, N₃, SR¹⁵,or OR²⁰, wherein R²⁰ is hydrogen, substituted lower alkyl, unsubstitutedlower alkyl, or C(═O)R¹⁴;

—CH(OH)(CH₂)_(j)R¹⁹;

—(CH₂)_(d)CHR²¹CO₂R^(16A), wherein d is 0-5, and R²¹ is hydrogen,CONR¹⁰R¹¹, or CO₂R¹⁶A, wherein R¹⁶A is the same as R¹⁶;

—(CH₂)_(d)CHR²¹CONR¹⁰R¹¹;

—CH═CH(CH₂)_(m)R²², wherein m is 0-4, and R²² is hydrogen, lower alkyl,CO₂R⁹, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, OR¹², or NR¹⁰R¹¹;

—CH═C(CO₂R^(16A))₂;

—C═—C(CH₂)_(m)R²²;

—SO₂NR²³R²⁴, wherein R²³ and R²⁴ independently are hydrogen, loweralkyl, or groups that form a heterocycle with the adjacent nitrogenatoms;

—OCO₂R^(13A), wherein R^(13A) is the same as R¹³; or

—OC(═O)NR¹⁰R¹¹;

R³ is hydrogen; lower alkyl; carbamoyl; amino; tetrahydropyranyl;hydroxyl; C(═O)H; aralkyl; lower alkanoyl; or CH₂CH₂R²⁵, wherein R²⁵ ishalogen, amino, di-lower alkylamino, hydroxyl, or hydroxysubstitutedlower alkylamino;

X is hydrogen; formyl; carboxyl; lower alkoxycarbonyl; loweralkylhydrazinocarbonyl; —CN; lower alkyl;

—C(═O)NR²⁶R²⁷, wherein R²⁶ and R²⁷ independently are hydrogen,unsubstituted lower alkyl, or unsubstituted aryl; or R²⁶ and R²⁷ arecombined with a nitrogen atom to form a heterocyclic group;

—CH(R³⁴)W, wherein R³⁴ is hydrogen or lower alkyl, and W is—N═CHN(alkyl)₂; guanidino; N₃; NR²⁸R²⁹, wherein R²⁸ or R²⁹ is hydrogenor lower alkyl, and the other is hydrogen, allyl, alkanoyl,aryloxycarbonyl, unsubstituted alkyl, or the residue of an α-amino acidin which the hydroxy group of the carboxyl group is excluded; —CO₂R⁹;—C(═O)NR¹⁰R¹¹; —S(═O)_(r)R³⁰, wherein R³⁰ is substituted orunsubstituted lower alkyl, aryl, or heteroaryl; or —OR³¹, wherein R³¹ ishydrogen, substituted or unsubstituted alkyl, or substituted orunsubstituted alkanoyl;

—CH═N—R³², wherein R³² is hydroxyl, lower alkoxy, amino, guanidino,ureido, imidazolylamino, carbamoylamino, or NR²⁶R^(27A) (wherein R^(26A)is the same as R²⁶ and R^(27A) is the same as R²⁷); or

—CH₂Q wherein Q is a sugar residue represented by

 wherein V represents hydrogen, methyl, ethyl, benzyl, acetyl, ortrifluoroacetyl;

Y is hydrogen; —OH; —OC(═O)R³³, wherein R³³ is alkyl, aryl, or amino;—OCH₂O-alkyl; —O-alkyl; aralkyloxy; or X and Y are combined as —X—Y—toform, —CH₂OCO₂— or —CH₂N(R^(16B))CO₂— (wherein R^(16B) is the same asR¹⁶);

A¹ and A² are hydrogen, or both are combined to represent O; or B¹ andB² are hydrogen, or both are combined to represent O; or apharmaceutically acceptable salt thereof; with the proviso that at leastone of A¹,A² or B¹,B² represents O; and with the further proviso thatboth X and Y are not simultaneously hydrogen.

Preferably, X is —C(═O)NR²⁶R²⁷, carboxyl, lower alkoxycarbonyl, formyl,lower alkyl, —CH₂OR³¹, —CH₂NR²⁸R²⁹, or —CH₂S(O)_(r)R³⁰. Preferably, R¹and R² are H. Preferably, R³ is hydrogen or a protecting group.Particularly preferred are Compounds VI, VII, VIII, X, XII, XIV, XV,XVI, XVII, XVIII, XIX, XXV, and XXVII, shown below:

In some embodiments of the invention, 3′-epimeric K252a derivatives areformulated into pharmaceutical compositions.

The invention also provides a method for inhibiting the activity of atyrosine kinase, for example, protein kinase C (PKC). The methodincludes contacting the tyrosine kinase with a compound of claim 1. Thetyrosine kinase can be in vivo or in vitro.

The invention also provides a method for inhibiting the phosphorylationof a tyrosine kinase by a second kinase. The method includes contactingthe second kinase with a compound of claim 1. The tyrosine kinase can bein vivo or in vitro.

The invention also provides a method for enhancing the function of acholinergic neuron. The method includes contacting the cholinergicneuron with a compound of claim 1. The cholinergic neuron can be in vivoor in vitro.

The invention also provides a method for enhancing the survival of acholinergic neuron. The method includes contacting the cholinergicneuron with a compound of claim 1. The cholinergic neuron can be in vivoor in vitro.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including definitions will control. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described below. The materials,methods, and examples are illustrative only and not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the synthesis of 3′-epimericindolocarbazoles VI and VII from the known indolocarbazole IIIa.

FIG. 2 is a drawing showing the synthesis of the 3′-epimericindolocarbazole X from the 3′-epimeric indolocarbazole VII, prepared asshown in FIG. 1.

FIG. 3 is a drawing showing the synthesis of 3′-epimeric indolocabazolesXIIa, XIV, XV, XVI, XVII, and XVIII from the intermediate ketone XIa.

FIG. 4 is a drawing showing the synthesis of 3′-epimericindolocarbazoles XIX from the intermediate epoxide IXa.

FIG. 5 is a drawing showing an alternate synthesis of the 3′-epimericindolocarbazoles XIX from the intermediate ketone XI.

FIG. 6 is a drawing showing the synthesis of the epimeric3′-hydroxyindolocarbazole XXVII from the intermediate ketone XI, andalternatively by epimerization of the known 3′-hydroxyindolocarbazoleXXVI.

FIG. 7 is a drawing showing the synthesis of a ring-brominated3′-epimeric indolocarbazole XXV from the corresponding 3′-epimericindolocarbazole XIV.

FIG. 8 is a graph summarizing data from experiments to determine theeffect of Compound XIV on survival of neurons in cultures enriched formotoneurons. Cell viability (as percent of control) is plotted againstconcentration of Compound XIV in the cell culture medium.

Epimeric K-252a derivatives of the invention display pharmacologicalactivities, including inhibition of tyrosine kinase activity, e.g.,inhibition of PKC or trk tyrosine kinase, which inhibition may be usefulin treatment of diseases, including cancer. Compounds of the inventionare useful for enhancing the function and/or survival of trophic factorresponsive cells, e.g., cholinergic neurons. Effects on neurons can bedemonstrated in assays including the following: (1) cultured spinal cordcholine acetyl-transferase (“ChAT”) assay; and (2) cultured basalforebrain neuron (“BFN”) ChAT activity assay.

ChAT is an enzyme in the biosynthetic pathway leading to acetylcholine.CHAT activity associated with a cholinergic neuron indicates that theneuron is functional. Neuron survival can be assayed by measuring uptakeand enzymatic conversion of a dye, e.g., calcein AM, by neurons.

Various neurological disorders are characterized by neuronal cells thatare injured, functionally comprised, undergoing axonal degeneration,dying, or at risk of dying. These disorders include: Alzheimer'sdisease, motor neuron disorders such as amyotrophic lateral sclerosis,Parkinson's disease; cerebrovascular disorders. such as stroke orischaemia, Huntington's disease, AIDS dementia, epilepsy, multiplesclerosis, peripheral neuropathies, disorders induced by excitatoryamino acids, and disorders associated with concussive or penetratinginjuries of the brain or spinal. cord.

Because they enhance trophic factor-induced activities of trophic factorresponsive cells, compounds of the invention can be used as therapeuticagents to enhance the function and/or survival of cells of neuronallineage in a mammal, e.g., a human. In particular, they are useful intreatment of disorders associated with decreased ChAT activity or injuryto spinal cord motoneurons.

Chemical Syntheses

Compounds of the invention can be prepared as described below (FIGS.1-7). The compounds can be prepared by starting with a suitablyprotected K-252a derivative. K-252a can be protected on the lactam amidenitrogen, e.g., as an acetate or as a silyl derivative.

Thionocarbonate IVa (FIG. 1) can be prepared from diol IIIa using aprocedure such as that described in U.S. Pat. No. 4,923,986. Treatmentof IVa with trimethylphosphite gives the exocyclic alkene V. Alkene Vcan be converted to (S)-methanol derivative VI using hydroborationconditions, or converted to (R)-diol VII using osmonium tetraoxide intetrahydrofuran (THF). In Compound VII, the configuration at the sugar3′-position is opposite to that reported as (S)-diol III in U.S. Pat.No. 4,923,986.

The (R)-epoxide IXa (FIG. 2) can be prepared by converting (R)-diol VIIto the tosyl intermediate VIII, followed by treatment with a base suchas sodium hydride or sodium hydroxide. Treatment of (R)-epoxide IXa withhydride reducing agents such as lithium triethyl borohydride gives thetertiary (S)-alcohol X after deprotection of the t-butyldimethyl silyl(TBDMS) group. Chiral alcohol derivatives such as compounds VI, VII or Xcan be further converted to ether derivatives by reaction with a baseand a halide or tosyl partner using conventional techniques. The alcoholderivatives also can be converted to ester derivatives by treatment withacid chlorides or anhydrides, or carbamates by reaction with anappropriate isocyanate by known procedures. Halide or sulfonatederivative of, for example, compounds VI or VII, can be displaced withvarious O, S, N, or C nucleophiles to yield a suitable derivative.

The preparation of 3′-(R)-K-252a XIV (FIG. 3) begins with ketone XI.Compound XIV differs from the natural K-252a isomer only at the 3′sugarposition. Treatment of ketone XIa with cyanide salts (NaCN, KCN,tetrabutylammonium cyanide, or TMSCN) gives a mixture of cyanohydrinsXII and XIII. The mixture of cyanohydrin isomers can be separated bychromatography or directly converted to ester XIV or amide XV using HClin methanol. 3′-epi-K-252a XIV can be hydrolysed to the hydroxy acid XVIusing a procedure such as that used for natural K-252a. See, e.g., J.Antibiot.39:1072, 1986. Acid XVI can be converted to a variety of esteror amide derivatives using similar procedures to those described forK-252a. See, e.g., U.S. Pat. Nos. 4,923,986; 5,461,146; and 5,654,427.Amide XV can be reduced to the corresponding methylamine derivative XVIIusing the procedure described for conversion of natural K-252a, and XVIIcan be used to prepare a number of methyl-amide and -urea derivatives.See, e.g., U.S. Pat. Nos. 4,923,986; 5,461,146; and 5,654,427.3′-epi-K-252a can be reduced to the aldehyde XVIII and condensed withvarious amines, hydrazines, or hydroxyl amines to form the correspondinganalogs. The aldehyde XVIII may be treated with various metal alkyl,arylalkyl, aryl, or heteroarylalkyl reagents, e.g.., Li, Mg, Zn or Cureagents, to form the corresponding alcohol addition products. AldehydeXVIII may be converted to functionalized olefins and their reducedproducts by treatment with phosphonium ylides (Quart Rev. 17:406, 1963;Angew Int. 16:423, 1977), phosphonates (Horner-Wadsworth-Emmonsreagents: Chem. Ber. 91:61, 1958; J. Am. Chem. Soc. 83:1733, 1961; Org.React. 25:73, 1977), silanes (J. Org. Chem. 33:780, 1968; Synthesis 384,1984) tellurium reagents (Tetrahedron Lett. 28:801, 1987) or boronreagents (Tetrahedron Lett. 24:635, 1983), followed by reduction of thealkene, e.g. by catalytic hydrogenaton. The alkene derived-from aldehydeXVIII can be converted to an epoxide and treated, for example, with anucleophile, as described for epoxide IX.

Epoxide IX (FIG. 4) can be treated with a variety of nucleophiles toform tertiary alcohols of structure XIX. The nucleophile can besubstituted. An alternative method (FIG. 5) to prepare epoxides andtertiary 3′-epi-OH configurations of the alcohols is to convert ketoneXI to an olefin of structure XX using a conventional olefinationreaction, e.g., as described for aldehyde XVIII.

The epoxide of structure XXI can be prepared asymmetrically using knownmethods. See, e.g., J. Org. Chem. 32:1363, Synthesis 89, 1986; 1967; J.Org. Chem. 60:3692, 1995; J. Am. Chem. Soc. 112:2801, 1990; J. Am. Chem.Soc. 116:6937, 1994; J. Org. Chem. 58:7615, 1993). Nucleophilic epoxideopening in a manner similar to that used with epoxide IX gives thesubstituted tertiary alcohol with the OH group in the 3′-epiconfiguration.

The known (R)-alcohol XXVI (FIG. 6) can be converted to (S)-alcoholXXVII using conventional methods for inversion of a secondary alcohol.See, e.g., Tetrahedron Lett. 34:6145, 1996; Synthesis Letters, 1995,336). Alternatively, XXVII can be prepared by treatment of epoxide XXIVwith a hydride reagent such as lithium triethyl borohydride. Ketone XI(FIG. 6) can be converted to triflate XXII followed by treatment withtributyltin hydride to give alkene XXIII.

Known methods used to prepare K252a derivatives with substitutents atpositions R₁ and R₂ can be employed to obtain the corresponding R₁ andR₂ substitutents on 3′-epi-K-252a. See, e.g., U.S. Pat. Nos. 4,923,986;5,461,146; and 5,654,427. For example, treatment of XIV (FIG. 7) withone equivalent of N-bromosuccinimide (NBS) yields the derivative XXV inwhich R₁ is Br. Two equivalents of NBS would give the derivative inwhich both R₁ and R₂ are Br.

Oxidation of (S)-methanol VI to an aldehyde or a carboxylic acidderivative can be achieved using appropriate oxidizing reagents (as.described in Oxidations in Organic Chemistry, American Chemical SocietyMonograph 186, ACS Washington DC 1990). The aldehyde or carboxylic acidderivatives can be further transformed using described procedures toprepare derivatives XVI and XVIII (FIG. 3).

Pharmaceutical Compositions

A compound of the invention can be administered. to a mammal, e.g., ahuman patient, as the sole active ingredient or in combination withother therapeutic agents. Compounds of the invention can be formulatedinto pharmaceutical compositions by admixture with pharmaceuticallyacceptable excipients and carriers. Such compositions can be formulatedfor any route of administration, e.g., parenteral, oral, nasal, ortopical. The composition can be administered in unit dosage form,following preparation by conventional methods. See, e.g., Remington'sPharmaceutical Sciences (Mack Pub. Co., Easton, Pa.). The amount andconcentration of the active ingredient can vary. The concentration willdepend upon factors such as the total dosage of the active ingredient,the chemical characteristics (e.g., hydrophobicity) of the compoundsemployed, the route of administration, the patient's age, the patient'sweight, and the condition being treated.

Compounds of the invention can be provided in an physiological buffersolution containing, e.g., 0.1 to 10% w/v compound for parenteraladministration. Typical dose ranges are from about 1 μg/kg to about 1g/kg of body weight per day; a preferred dose range is from about 0.01mg/kg to 100 mg/kg of body weight per day, and preferably about 0.1 to20 mg/kg once to four times per day.

The invention includes pharmaceutically acceptable salts of 3′-epimericK252a derivatives. Pharmaceutically acceptable salts includepharmaceutically acceptable acid addition salts, metal salts, ammoniumsalts, organic amine addition salts, and amino acid addition salts. Acidaddition salts include inorganic acid addition salts such ashydrochloride, sulfate and phosphate, and organic acid addition saltssuch as acetate, maleate, fumarate, tartrate, citrate and lactate.Examples of metal salts are alkali metal salts such as lithium salt,sodium salt and potassium salt, alkaline earth metal salts such asmagnesium salt and calcium salt, aluminum salt, and zinc salt. Examplesof ammonium salts are ammonium salt and tetramethylammonium salt.Examples of organic amine addition salts are salts with morpholine andpiperidine. Examples of amino acid addition salts are salts withglycine, phenylalanine, glutamic acid and lysine.

As used herein, “lower alkyl” means an alkyl group with 1 to 6 carbons.As used herein, “aryl” (alone or in terms such as arylcarbonyl andarylaminocarbonyl) means a group having 6 to 12 carbon atoms, in asingle ring, or two fused rings. Examples of aryl groups are phenyl,biphenyl and naphthyl. A heteroaryl group contains at least one heteroatom. Preferably, the hetero atom is O, S, or N. Examples of heteroarylgroups are pyridyl, pyrimidyl, pyrrolyl, furyl, thienyl, imidazolyl,triazolyl, tetrazolyl, quinolyl, isoquinolyl, benzoimidazolyl, thiazolyland benzothiazolyl. A substituted alkyl group has 1 to 3independently-selected substituents. Preferred substituents for alkylgroups are hydroxy, lower alkoxy, substituted or unsubstitutedarylalkoxy-lower alkoxy, substituted or unsubstitutedheteroarylalkoxy-lower alkoxy, halogen, carboxyl, lower alkoxycarbonyl,nitro, amino, mono- or di-lower alkylamino, dioxolane, dioxane,dithiolane, and dithione. A substituted aryl, heteroaryl or arylalkylgroup has 1 to 3 independently-selected substituents. Preferredsubstituents are lower alkyl, hydroxy, lower alkoxy, carboxy, loweralkoxycarbonyl, nitro, amino, mono- or di-lower alkylamino, and halogen.

As used herein, “cholinergic neuron” means a neuron that usesacetylcholine as a neurotransmitter. Examples of cholinergic neurons arebasal forebrain neurons, striatal neurons, and spinal cord neurons. Asused herein, “sensory neuron” means a neuron responsive to anenvironmental stimulus such as temperature or movement. Sensory neuronsare found in structures including skin, muscle and joints. A dorsal rootganglion neuron is an example of a sensory neuron. As used herein,“trophic factor-responsive cell” means a cell to which a trophic factorbinds. Trophic factor-responsive cells include cholinergic neurons,sensory neurons, monocytes and neoplastic cells.

The invention is further illustrated by the following examples. Theexamples are not to be construed as limiting the scope or content of theinvention in any way.

EXPERIMENTAL EXAMPLES

Inhibition of Tyrosine Kinase Activity

Epimeric K252a derivatives were tested for inhibition of kinase activityof baculovirus-expressed human trkA cytoplasmic domain using anELISA-based assay as described by Angeles et al. (Anal. Biochem.236:49-55, 1996). A 96-well microtiter plate was coated with substratesolution (recombinant human phospholipase C-γ/glutathione S-transferasefusion protein; Rotin et al., EMBO J., 11:559-567, 1992). Inhibition wasmeasured in 100 ml assay mixtures containing 50 mM Hepes, pH 7.4, 40 μMATP, 10 mM MnCl₂, 0.1% BSA, 2% DMSO, and various concentrations ofinhibitor. The reaction was initiated by addition of trkA kinase andallowed to proceed for 15 minutes at 37° C. An antibody tophosphotyrosine (UBI) was then added, followed by a secondaryenzyme-conjugated antibody, alkaline phosphatase-labelled goatanti-mouse IgG (Bio-Rad).

The activity of the bound enzyme was measured using an amplifieddetection system (Gibco-BRL). Inhibition data were analyzed using thesigmoidal dose-response (variable slope) equation in GraphPad Prism. Theconcentration that gave 50% inhibition of kinase activity was referredto as IC₅₀. Results are summarized .in Table 1.

TABLE 1 Inhibition of trkA Kinase Activity by 3'-Epimeric K-252aDerivatives trk IC₅₀ Compound (nM) VI 2 VII 2 X 2 XIV 1.4 XV 21 XXIX 7(Control)

Inhibition of NGF-stimulated trk Phosphorylation

The inhibition of NGF-stimulated phosphorylation of trk by selectedepimeric K-252a derivatives was measured using a procedure modified fromthat described in U.S. Pat. No. 5,516,771. NIH3T3 cells transfected withtrkA were grown in 100 mm dishes. Subconfluent cells wereserum-starved-by replacing media with serum-free 0.05% BSA-DMEMcontaining-compound (1-100 nM) or DMSO (added to controls) for one hourat 37° C. NGF (Harlan/Bioproducts for Science) was then added to thecells at a concentration of 10 ng/ml for 5 minutes. Cells were lysed inbuffer containing detergent and protease inhibitors. Clarified celllysates were normalized to protein using BCA method andimmunoprecipitated with anti-trk antibody.

Polyclonal anti-trk antibody was prepared against a peptidecorresponding to the 14 amino acids at the carboxy terminus of trk(Martin-Zanca et al., Mol. Cell. Biol 9:24-33, 1989). The immunecomplexes were collected on Protein A Sepharose beads (Sigma), separatedby SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred toa polyvinylidene difluoride (PVDF) membrane. The membrane wasimmunoblotted with anti-phosphotyrosine antibody (UBI), followed byincubation with horseradish peroxidase coupled goat anti-mouse IgG(Bio-Rad).. Phosphorylated proteins were visualized using ECL(Amersham).

The area of the trk protein band was measured and compared toNGF-stimulated control. The inhibition scoring system used, based onpercent decrease in trk protein band, was as follows: 0=no decrease;1=1-25%; 2=26-49%; 3=50-75%; 4=76-100%.

The trk inhibition data (Table 2) revealed that the 3′epi-OH isomerswere more potent for inhibiting trk in a whole cell preparation than thecorresponding natural isomers. 3′-epi-K-252a. (XIV) displayed an IC₅₀ of<10 nM, whereas K-252a displayed an IC₅₀ of approximately 50 nM.Compound X showed a complete inhibition of trkA at <50 nM, and an IC₅₀of <10 nM in cells. The natural isomer XXIX did not show completeinhibition at 100 nM. Diol VII displayed greater potency than thenatural isomer III for trkA inhibition in NIH3T3 cells.

TABLE 2 Effects of 3'-Epimeric K-252 Derivatives on NGF-stimulated trkAPhosphorylation in NIH3T3 Cells Inhibition Score Compound 1 nM 10 nM 50nM 100 nM K-252a 1 2 3 3 (Control) XIV 2 4 4 4 VII 1 2 4 4 III 1 2 3 4(Control) X 2 3 4 4 XXIX 2 3 3 3 (Control)

Inhibition of VEGF Receptor Kinase Activity

3′-Epimeric K-252a derivatives were tested for inhibition of the kinaseactivity of baculovirus-expressed VEGF receptor kinase domain, using theprocedure described above. The kinase reaction mixture, consisting of 50mM Hepes, pH 7.4, 40 μM ATP, 10 mM MnCl₂, 0.1% BSA, 2% DMSO, and variousconcentrations of inhibitor, was transferred to PLC-γ/GST-coated plates.VEGFR kinase was added and the reaction was allowed to proceed for 15min. at 37° C. Phosphorylated product was detected byanti-phosphotyrosine antibody (UBI). A secondary enzyme-conjugatedantibody was used to capture the antibody-phosphorylated PLC-γ/GSTcomplex. The activity of the bound enzyme was measured by an amplifieddetection system (Gibco-BRL). Inhibition data were analyzed using thesigmoidal dose-response (variable slope) equation in GraphPad Prism.Results are summarized in Table 3.

TABLE 3 Inhibition of VEGF Receptor Kinase Activity by 3'-EpimericK-252a Derivatives VEGFR kinase IC₅₀ Compound (nM) VI 7 VII 8 X 17 XIV19 XXIX 146 (Control)

Inhibition of Protein Kinase C Activity

Protein kinase C activity was measured using the Millipore MultiscreenTCA in-plate assay (Pitt et al., J. Biomol. Screening 1:47-51, 1996).Assays were performed in 96-well Multiscreen-DP plates (Millipore). Each40-ml assay mixture contained 20 mM Hepes, pH 7.4, 10 mM MgCl₂, 2.5 mMEGTA, 2.5 mM CaCl₂, 80 mg/ml phosphatidyl serine, 3.2 mg/ml diolein, 200mg/ml histone H-1 (Fluka), 5 mM [γ-³²P]ATP, 1.5 ng protein kinase C(UBI; mixed isozymes of a, b, g), 0.1% BSA, 2% DMSO, and 3′-epimericK-252a derivative. The reaction was allowed to proceed for 10 min at 37°C. The reaction was quenched with ice cold 50% trichloroacetic acid(TCA). The plates were equilibrated for 30 min at 4° C., then washedwith ice cold 25% TCA. Scintillation cocktail was added to the plates,and the radioactivity was determined using Wallac MicroBeta 1450 PLUSscintillation counter. The IC₅₀ values were calculated by fitting thedata to the sigmoidal dose-response (variable slope) equation inGraphPad Prism. The results are summarized in Table 4.

TABLE 4 Inhibitory Effects of 3'-Epimeric K-252a Derivatives on ProteinKinase C Activity PKC Compound IC₅₀ (nM) VI 95 VII 79 X >1000 XIV 114XXIX 310 (Control)

Enhancement of Spinal Cord ChAT Activity

ChAT was employed a biochemical marker for functional cholinergicneurons. CHAT activity has been used to study the effects ofneurotrophins (e.g., NGF or NT-3) on the survival and/or function ofcholinergic neurons. The ChAT assay also has been used as an indicationof the regulation of ChAT levels within cholinergic neurons.

3′-Epimeric K-252a derivatives increased ChAT activity in thedissociated rat embryonic spinal cord culture assay (Table 5). CompoundXVII increased ChAT activity 195% over control cultures (not treatedwith the epimeric K-252a derivative) after allowing a 2-3 hour platingperiod for cells to attach to control tissue culture wells. In theseassays, a compound was directly added to a dissociated spinal cordculture. Compounds which increased CHAT activity at least 120% of thecontrol activity were considered active. Increased ChAT activity wasobserved after a single application of a selected epimeric K-252aderivative. Results are summarized in Table 5.

TABLE 5 Enhancement of Spinal Cord ChAT Activity by 3'-Epimeric K-252aDerivatives Spinal Cord ChAT % control Activity at Maximal Compound 50nM Activity VI <120 125 VII <120 122 X <120 127 XIV 147 195 XV — 129

Fetal rat spinal cord cells were dissociated, and experiments wereperformed, essentially as described by Smith et al., J. Cell Biology101:1608-1621, 1985), and Glicksman et al., J. Neurochem. 61:210-221,1993). Dissociated cells were prepared from spinal cords dissected fromrats (embryonic day 14-15) by conventional trypsin dissociationtechniques. Cells were plated at 6×10⁵ cells/cm² on poly-1-ornithinecoated plastic tissue culture wells in serum-free N2 medium supplementedwith 0.05% bovine serum albumin (BSA) (Bottenstein et al., Proc. Natl.Acad. Sci. USA 76:514-517, 1979). Cultures were incubated at 37° C. in ahumidified atmosphere of 5% CO₂/95% air for 48 hours. ChAT activity wasmeasured after 2 days in vitro using a modification of the Fonnumprocedure (Fonnum, J. Neurochem. 24:407-409, 1975) according toMcManaman et al. (Developmental Biology 125:311-320, 1988) and Glicksmanet al. (supra).

Survival Assay Using Rat Spinal Cord Motoneurons

Selected 3′-epimeric K-252a derivatives were assayed forsurvival-enhancing activity in rat spinal cord motoneurons. Compound XVIsignificantly enhanced survival of spinal cord motoneurons (FIG. 8).

Spinal cords were dissected from Sprague-Dawley rat fetuses (CharlesRiver Laboratories, Wilmington, Mass.) of embryonic age (E) 14.5-15.Cells from only the ventral portion of the spinal cord were dissociated,and further enriched for motoneurons by centrifugation on a 6.5% stepmetrizamide gradient, and were analyzed for purity by staining with lowaffinity neurotrophin receptor antibody (IgG-192, Boehringer-Mannheim).Cells were seeded onto 96-well plates previously coated withpoly-1-ornithine and laminin (5 ug/ml each) at a density of 6×10⁴cells/cm² in chemically defined serum-free N2 medium (Bottenstein andSato, 1979). To distinguish attachment from survival effects,3,9-bis[(ethylthio)methyl]-K-252a (Kaneko et. al., J. Med. Chem40:1863-1869, 1997) was added to cultures after an initial attachmentperiod of 1-3 hours.

Neuronal survival was assessed after 4 days by calcein AM (MolecularProbes, Eugene, Oreg.) in a fluorimetric viability assay (Bozyczko-Coyneet. al., J. Neurosci. Meth. 50:205-216, 1993). Culture medium wasserially diluted in Dulbeccos phosphate buffered saline (DPBS). A finalconcentration of 6 uM calcein AM stock was added to each well. Theplates were incubated for 30 min at 37° C., followed by serial dilutionwashes in DPBS. The fluorescent signal was read using a plate-readingfluorimeter (Cytofluor 2350) (excitation=485 nm; emission=538 nm). Foreach plate, mean background derived from wells receiving calcein AM, butcontaining no cells, was subtracted from all values. Linearity of thefluorescence signal was verified for the concentration and incubationtime for the range of cell densities. Microscopic counts of neuronscorrelated directly with relative fluorescence values.

Preparation of Compound V

Compound IVb (U.S. Pat. No. 4,923,986) was dissolved intrimethylphosphite (2 mL) and heated to reflux for 3 hours. The reactionmixture was cooled to room temperature and flushed through a flashsilica gel column using chloroform-methanol (20:1) to removetrimethylphosphite. The product was purified by flash chromatography(silica gel; ethyl acetate:hexane; 1:1) to give compound V as a paleyellow solid (15 mg, 95% yield). MS (ESI⁺): m/e 406 (M+H)⁺, ¹H NMR(CDCl₃, 300 MHz): δ2.62 (s, 3H), 2.85 (d, 1H), 3.37-3.45 (m, 1H), 4.95(s, 1H), 5.00 (s, 2H), 5.09 (s, 1H), 6.29 (s, 1H), 6.90 (d, 1H),7.33-7.53 (m, 5H), 7.91 (d, 1H), 9.41 (d, 1H).

Preparation of Compound VI

To a stirred solution of compound V (161 mg, 0.397 mmols) in THF (8mL)at 0° C. under nitrogen was added BH₃ THF (1.59 mL of a 1M solution,1.59 mmol). The reaction mixture was stirred for 30 min at 0° C. andthen warmed to room temperature overnight. The mixture was recooled to0° C. and 10% NaOH (0.1 mL) was added, with vigorous evolution of gas.Hydrogen peroxide (80 mL) was then added dropwise. After stirring at 0°C. for 30 min, the reaction was diluted with ethyl acetate (15 mL) andwashed with water (3×10 mL). The organic layer was dried over sodiumsulfate, filtered and concentrated in vacuo to a light green solid. Theproduct was purified by chromatography (silica gel: hexane:ethylacetate; 1:1) to give compound VI as a white solid (0.12g, 71% yield).MS (ESI⁺): m/e 424 (M+H)⁺; ¹H NMR (CDCl₃, 300 MHz): δ2.54 (s, 3H), 2.51(m, 1H), 2.99-3.01 (m, 2H), 3.47 (m, 1H), 3.8 (m, 1H), 5.049 (s, 2H),6.21 (broad 2, 1H), 6.98 (m, 1H), 7.13-7.49 (m, 6H), 7.94-8.02 (m, 2H) ,9.34 (d, 1H).

Preparation of Compound VII

To a stirred solution of compound Va (TBDMS-V) (350 mg, 0.673 mmols) inTHF (10 mL) at room temperature under nitrogen was added pyridine (0.435mL, 5.39 mmol) followed by osmium tetroxide (6.73 mL, 0.673 mmol, 0.1 Min CCl₄). The reaction mixture was stirred at room temperature 36 h.During this time, the mixture changed color from yellow to orange-brown.Aqueous sodium bisulfite (30 mL) was added to the reaction mixture andthe reaction was stirred for 30 min. The reaction mixture was extractedwith EtOAc (2×20 mL), dried over sodium sulfate, filtered andconcentrated in vacuo to give a light brown film. The mixture waspurified by flash chromatography on silica gel using ethyl acetate toyield compound VIIa (TBDMS-VII) as a yellow solid (280 mg, 76% yield).MS (ESI⁺): m/e 544 (M+H)⁺, ¹H NMR (CDCl₃, 300 MHz): δ0.56 (d, 6H), 1.079(s, 9H), 2.04 (dd, 1H), 2.12 (broad s, 1H), 2.40 (s, 3H), 2.86 (dd, 1H),3.52 (broad s, 3H), 4.99 (s, 2H), 6.98 (dd, 1H), 7.32 (t, 1H), 7.39-7.46(m, 4H), 7.97 (dd, 2H), 9.35 (d, 1H).

To a flask containing methanol (2 mL) at 0° C. under nitrogen was addedacetyl chloride (4 drops). Compound VIIa (40 mg, 0.072 mmols) inmethanol (1 mL) was added dropwise to the solution of methanolic HCl.The reaction mixture was stirred at 0° C. for 1 hour then was warmed toroom temperature overnight. The solvent was removed in vacuo to givecompound VII as a tan solid (21 mg, 66% yield). MS (ESI⁺): m/e 440(M+H)⁺; ¹H NMR (CDCl₃, 300 MHz): δ2.052 (dd, 1H), 2.43 (s, 3H), 2.90(dd, 1H), 3.57 (s, 1H), 3.61 (s, 2H), 5.04 (s, 2H), 6.28 (s, 1H), 7.02(dd, 1H), 7.33-7.54 (m, 6H), 7.95 (d, 1H), 8.02 (d, 1H), 9.32 (d, 1H).

Preparation of Compound X

To a stirred solution of intermediate VIIa (FIG. 2; 0.23g, 0.415 mmols)in THF (10 mL) at 0° C. under nitrogen was added triethylamine (57.9 ml,0.415 mmols), DMAP (25.4 mg, 0.208 mmol) and p-toluenesulfonyl chloride(79.1 mg, 0.415 mmol). The reaction mixture was stirred at 0° C. for 1hour. It was then slowly warmed to room temperature overnight. Thereaction mixture was warmed for 1 hour, while monitoring by thin layerchromatography (hexane:ethyl acetate, 2:1). The reaction mixture wasdiluted with ethyl acetate (30 mL) and washed with water (3×15 mL). Theorganic phase was dried over sodium sulfate, filtered, and concentratedin vacuo to yield the tosyl intermediate VIIIa as a yellow film. Thereaction mixture was further purified by flash chromatography on silicagel using hexane:ethyl acetate (2:1) to yield a light yellow film (0.16g, 55% yield). MS (APCI): m/e 708 (M+H)⁺; ¹H NMR (CDCl₃, 300 MHz): δ0.57(d, 6H), 1.08 (s, 9H), 2.01 (dd, 1H), 2.33 (s, 3H), 2.42 (s, 3H), 3.88(dd, 1H), 3.86 (dd, 2H), 4.98 (s, 2H), 6.97 (dd, 1H), 7.14 (d, 2H),7.24-7.49 (m, 7H), 7.75 (d, 1H) 7.91 (d, 1H) , 9.35 (d, 1H)

To a stirred solution of intermediate VIIIa (0.14 g, 0.198 mmols) in THF(5 mL) at 0° C. under nitrogen was added sodium hydride (15.8 mg, 0.395mmol). Vigorous evolution of gas was observed, and the reaction mixturebecame cloudy. Additional sodium hydride (2 eq) was added and thecontents of the flask were stirred for an additional 2 hours, thenwarmed gently for 4 h. The reaction mixture was then cooled to 0° C. andquenched with water. The reaction mixture was diluted with ethyl acetateand washed with water and brine. The organic phase was dried over sodiumsulfate, filtered and concentrated in vacuo to give Compound IXa as ayellow film (100.2 mg, 95% yield). MS (APCI: m/e 536 (M+H)⁺; ¹H NMR(CDCl₃, 300 MHz): δ0.56 (d, 6H), 1.08 s, 9H), 2.32-2.38 (m, 4H), 2.57(d, 2H), 3.01 (dd, 1H), 4.99 (s, 2H), 7.01 (d, 1H), 7.33-7.56 (m, 5H),7.73 (d, 1H), 7.94 (d, 1H), 9.46 (d, 1H).

To a stirred solution of Compound IXa (100 mg, 0.187 mmol) in THF (5 mL)at 0° C. under nitrogen was added lithium triethyl-borohydride (0.37 mLof 1M solution in THF, 0.374 mmol) dropwise, with evolution gas.Additional lithium triethylborohydride (2 eq) was added at 0° C. and thereaction was stirred at 0° C. for 30 minutes and then was warmed to roomtemperature. The reaction mixture was cooled to 0° C. and quenched withwater, diluted with ethyl acetate and washed with water and brine. Theorganic phase was dried over magnesium sulfate filtered and concentratedin vacuo. The reaction mixture was purified by flash chromatography onsilica gel using hexane:ethyl acetate (1:1) to yield Xa (R=TBDMS) as apale yellow film. To a stirred solution of Compound Xa in methanol at 0°C. under nitrogen was added a solution made from acetyl chloride (5drops) in methanol. The reaction mixture was stirred at 0° C. for 30minutes, then was warmed to room temperature overnight. The solvent wasremoved in vacuo leaving a yellow solid which was purified by silica gelchromatography (hexane:ethyl acetate;1:1) to give Compound X (30 mg,42%). MS (ESI⁺): m/e 424 (M+H)⁺, ¹H NMR (CDCl₃, 300 MHz): δ1.39 (s, 3H),2.29 (dd, 1H), 2.37 (s, 3H), 2.91 (dd, 1H), 5.05 (s, 2H), 6.19 (s, 1H),6.97 (t, 1H), 7.32-7.50 (m, 5H), 7.78 (d, 1H), 7.95 (d,1H), 9.33 (d,1H).

Preparation of Compounds XIV and XV

To a stirred solution of ketone XI (U.S. Pat. No. 4,923,986) (FIG. 5;451 mg, 1.11 mmols) in a CH₂Cl₂-dioxane mixture (6 mL; 5:1) undernitrogen was added tetrabutylammonium cyanide (740 mg, 2.77 mmols) andacetic acid (95 mL, 1.66 mmols) at room temperature. The dark reactionmixture was stirred for 24 hours, and then concentrated in vacuo. Thedark oil was dissolved in ethyl acetate (20 mL) and dioxane (2 mL) andwashed with water (3×10 mL) and brine (1×10 mL). The organic phase wasdried over magnesium sulfate, filtered, and concentrated in vacuo to abrown solid. The HPLC analysis showed the presence of two cyanohydrinintermediates, XIIa and XIIIb.

To a flask containing methanol (4 mL) was added HCl(g) for 10 minutes. Asolution of the crude cyanohydrin mixture from step 1 (450 mg, 1.04mmols) in methanol:dioxane (2:1, 3 mL) was added to the HCl in methanolsolution at 0° C. The reaction mixture was sealed and stirred at 0° C.for 2 hours, then was placed in a refrigerator for 48 h. The flask waswarmed to room temperature and 6 N HCl was added carefully. The mixturewas stirred for 30 minutes and then concentrated to dryness. The residuewas dissolved in 50% methanol:water and a precipitate formed whilestirring overnight at room temperature. The reaction mixture wasconcentrated to dryness. The residue was purified by flashchromatography on silica gel using hexane:ethyl acetate (1:1) to yieldcompound XIV as an off-white solid. MS (ESI⁺): m/e 468 (M+H)⁺; ¹H NMR(CDCl₃, 300 MHz) δ2.416 (s, 3H, 2.77 (dd, 1H), 2.91 (s, 3H), 2.952 (dd,1H), 4.99 (s, 2H), 7.13 (dd, 1H), 7.33 (t, 1H), 7.44 (dd, 2H), 7.64 (t,2H), 7.98 (d, 1H), 9.16 (d, 1H). The column was eluted with ethylacetate to obtain the amide XV as a light orange product (13 mg). MS(ESI): m/e 453 (M+H)⁺; ¹H NMR (DMSO-d₆, 300 MHz) δ2.33 (s, 3H), 2.87 (m,2H), 4.94 (s, 2H), 6.58 (s, 1H), 7.19-7.64 (m, 6H), 7.81 (m, 3H), 7.96(d, 1H), 8.59 (s, 1H), 9.17 (d, 1H).

Preparation of Compound XXIII

To a stirred solution of ketone XIa (FIG. 6; R=TBDMS) (95.4 mg, 0.183mmol) in THF (5 mL) at −78° C. under nitrogen was added lithiumdiethylamide (0.12 mL, 1.5 M solution in cyclohexane). The reactionmixture stirred at −78° C. for 30 min. A solution ofN-phenyltrifluoromethanesulfonimide (71.9 mg, 0.201 mmol) in THF (1.5mL) was added dropwise to the reaction mixture and the mixture wasstirred at −78° C. for 30 min. The reaction mixture was allowed towarmed to 0° C., stirred for 1 hour, then warmed to room temperatureovernight. The reaction was quenched with ammonium chloride (sat. aq. 2mL), diluted with ethyl acetate and washed with water. The mixture waspurified by flash chromatography on silica gel using hexane:ethylacetate (2:1) to give compound XXIIa (R=TBDMS) as an off-white solid (66mg, 61% yield) . MS (ESI⁺): m/e 654 (M+H)⁺, ¹H NMR (CDCl₃, 300 MHz):δ0.58 (s, 6H), 1.12 (s, 9H), 2.66 (s, 3H), 5.02 (dd, 2H), 6.14 (s, 1H),7.31-7.62 (m, 6H), 7.83 (d, 1H), 7.98 (d, 1H), 9.44 (d, 1H).

To a stirred solution of the product from step 1 (compound XXIIa) (75mg, 0.115 mmol) in THF (10 mL) was added lithium chloride (14.6 mg,0.345 mmol) and tetrakis-(triphenylphosphine)palladium (0) (2.6 mg,0.0023 mmol). Tributyltin hydride (37 mL, 0.139 mmol) was added dropwiseand the contents were heated to 60° C. The reaction mixture was heatedfor 4 h, during which time the color of the reaction changed from yellowto red-black. The reaction was concentrated in vacuo and purified byflash chromatography on silica gel using hexane:ethylacetate (2:1). Twoproducts were isolated: TBDMS-protected XXIIIa product contaminated withtributyltin (60 mg) and deprotected product XXIII (10 mg, 22%). MS(ESI): m/e 392 (M+H)⁺, ¹H NMR (CDCl₃, 300 MHz): δ2.65 (s, 3H), 4.978 (s,2H), 6.19 (d, 1H), 6.28 (d, 1H), 6.41 (s, 1H), 7.26-7.62 (m, 5H),7.65-7.69 (m, 1H), 7.87 (dd, 2H) , 9.39 (d, 1H).

Compound XXIII displayed the following IC₅₀ values in the assaysdescribed above: inhibition of trkA kinase, 4 nM; inhibition of VEGFreceptor kinase, 25 nM, and inhibition of Protein Kinase C, >1000 nM.

Preparation of Compound XXV

To a stirred solution of compound XIV (30.4 mg, 0.065 mmols) in THF (5mL) at room temperature under nitrogen was added N-bromosuccinimide(11.6 mg, 0.065 mmols) in one portion. The reaction mixture was lightorange in color initially and gradually turned light purple. Thereaction mixture stirred at room temperature overnight. The solvent wasremoved in vacuo and the solid purified by flash chromatography onsilica gel using hexane-ethyl acetate (2:1). This yielded Compound XXVas an off-white solid (31.2 mg, 88% yield). MS (ESI): m/e 547 (M+H)+, 1HNMR (CDCl_(3, 300) MHz): δ2.42 (s, 3H), 2.77-2.83 (m, 1H), 2.86 (3H, s)31.66 (dd, 1H), 4.105 (s, 1H), 5.07 (s, 2H), 6.39 (s, 1H), 7.01 (dd,1H), 7.95 (d, 1H), 7.58 (d, 1H), 7.34-7.57 (m, 4H) , 9.50 (s, 1H) .

Preparation of Compound XXIX

Method A

To a stirred solution of epoxide XXVIII (U.S. Pat. No. 4,923,986,compound I-27) (90.1 mg, 0.152 mmol) in THF (4 mL) at 0° C. undernitrogen was added lithium triethylborohydride (0.455 mL of a 1M in THFsoln., 0.455 mmols) dropwise. The reaction mixture was stirred at 0° C.for 1 hour then allowed warmed to room temperature overnight. Themixture was cooled to 0° C. and quenched by the slow addition ofmethanol. Stirring was continued at 0° C. for 15 min, after which timethe reaction mixture was warmed to room temperature. The solvent wasremoved in vacuo. This yielded a yellow oil. The oil was purified byflash chromatography on silica using ethyl acetate:hexane (1:1). Thisyielded Compound XXIX as a white solid (75 mg, 83 % yield). MS (ESI):m/e 424 (M+H)⁺; ¹H NMR (CDCl₃, 300 MHz): δ1.69 (s, 3H), 1.99 (s, 3H),2.86 (dd, 1H), 3.03 (dd, 1H), 4.37 (m, 3H), 4.93 (s, 2H), 6.43 (t, 1H),6.95 (d, 1H), 7.03 (t, 1H), 7.18 (t, 1H) 7.44 (t, 1H), 7.79 (d, 1H),7.99 (d, 1H), 8.69 (d, 1H).

Method B

To a stirred solution of ketone XI (FIG. 3, R=H) (212 mg, 0.41 mmols) inTHF (6 mL) at 0° C. under nitrogen was added methylmagnesium iodide(0.27 mL, 0.82 mmol) dropwise. The reaction mixture stirred at 0° C. for1 hour then was warmed to room temperature overnight. The mixture wasthen heated to reflux for 24 h then cooled to room temperature. Thereaction was quenched with ammonium chloride (sat. aq.), diluted withethyl acetate (20 mL) and washed with water (3×10 mL). The organic phasewas dried over sodium sulfate, filtered, and concentrated in vacuo to ayellow residue. The product was purified by flash chromatography onsilica gel using hexane:ethyl acetate (1:1) to give compound XXIX as atan solid (0.11 g, 50% yield). The ¹H NMR and mass spectrometry datawere consistent with the product obtained from Method A.

Other embodiments are within the following claims.

We claim:
 1. A method of treating a neurological disorder comprisingadministering to a patient an effective amount of a compound of thefollowing formula:

wherein: R¹ and R² independently are: hydrogen; lower alkyl; halogen;acyl; nitro; sulfonic acid; —CH═NR⁴, wherein R⁴ is guanidino,heterocyclic, or —NR⁵R⁶, wherein one of R⁵ and R⁶ is hydrogen or loweralkyl, and the other of R⁵ and R⁶ is hydrogen, lower alkyl, acyl, aryl,heterocyclic, carbamoyl or lower alkylaminocarbonyl; —NR⁵R⁶; —CH(SR⁷)₂,wherein R⁷ is lower alkyl or alkylene; —(CH₂)_(j)R⁸, wherein j is 1-6,and R⁸ is halogen, substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl or N₃; —CO₂R⁹, wherein R⁹ ishydrogen, substituted lower alkyl, unsubstituted lower alkyl,substituted aryl, unsubstituted aryl, substituted heteroaryl, orunsubstituted heteroaryl; —C(═O)NR¹⁰R¹¹, wherein R¹⁰ and R¹¹independently are hydrogen, substituted lower alkyl, unsubstituted loweralkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted aralkyl, unsubstituted aralkyl,lower alkylaminocarbonyl, or lower alkoxycarbonyl, or R¹⁰ and R¹¹ arecombined with a nitrogen atom to form a heterocyclic group; —OR¹²,wherein R¹² is hydrogen, substituted lower alkyl, unsubstituted loweralkyl, substituted aryl, unsubstituted aryl or —C(═O)R¹³, wherein R¹³ ishydrogen, NR¹⁰R¹¹, substituted lower alkyl, unsubstituted lower alkyl,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted aralkyl, or unsubstituted aralkyl;—NR¹⁰R¹¹; —C(═O)R¹⁴, wherein R¹⁴ is hydrogen, lower alkyl, substitutedaryl, unsubstituted aryl, substituted heteroaryl, or unsubstitutedheteroaryl; —S(═O)_(r)R¹⁵, wherein r is 0 to 2, and R¹⁵ is hydrogen,substituted lower alkyl, unsubstituted lower alkyl, substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,substituted aralkyl, unsubstituted aralkyl, thiazolinyl,—(CH₂)_(a)C(═O)NR¹⁰R¹¹ or (CH₂)_(a)CO₂R¹⁶, wherein a is 1 or 2, and R¹⁶is hydrogen or lower alkyl; —OR¹⁷, wherein R¹⁷ is hydrogen, lower alkyl,or —C(═O)R¹⁸, wherein R¹⁸ is substituted lower alkyl, unsubstitutedlower alkyl, substituted aryl, or unsubstituted aryl;—C(═O)(CH₂)_(j)R¹⁹, wherein R¹⁹ is hydrogen, halogen, NR¹⁰R¹¹, N₃, SR¹⁵,or OR²⁰, wherein R²⁰ is hydrogen, substituted lower alkyl, unsubstitutedlower alkyl, or C(═O)R¹⁴; —CH(OH)(CH₂)_(j)R¹⁹;—(CH₂)_(d)CHR²¹CO₂R^(16A), wherein d is 0-5, and R²¹ is hydrogen,CONR¹⁰R¹¹, or CO₂R^(16A), wherein R^(16A) is the same as R¹⁶;—(CH₂)_(d)CHR²¹CONR¹⁰R¹¹; —CH═CH(CH₂)_(m)R²², wherein m is 0-4, and R²²is hydrogen, lower alkyl, CO₂R⁹, substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, OR¹², or NR¹⁰R¹¹;—CH═C(CO₂R^(16A))₂; —C≡C(CH₂)_(m)R²²; —SO₂NR²³R²⁴, wherein R²³ and R²⁴independently are hydrogen, lower alkyl, or groups that form aheterocycle with the nitrogen atoms; —OCO₂R^(13A), wherein R^(13A) isthe same as R¹³; or —OC(═O)NR¹⁰R¹¹; R³ is hydrogen; lower alkyl;carbamoyl; amino; tetrahydropyranyl; hydroxyl; C(═O)H; aralkyl; loweralkanoyl; or CH₂CH₂R²⁵, wherein R²⁵ is halogen, amino, di-loweralkylamino, hydroxyl, or hydroxysubstituted lower alkylamino; X ishydrogen; formyl; carboxyl; lower alkoxycarbonyl; loweralkylhydrazinocarbonyl; —CN; lower alkyl; —C(═O)NR²⁶R²⁷, wherein R²⁶ andR²⁷ independently are hydrogen, unsubstituted lower alkyl, orunsubstituted aryl; or R²⁶ and R²⁷ are combined with a nitrogen atom toform a heterocyclic group; —CH(R³⁴)W, wherein R³⁴ is hydrogen or loweralkyl, and W is —N═CHN(alkyl)₂; guanidino; N₃; NR²⁸R²⁹, wherein R²⁸ orR²⁹ is hydrogen or lower alkyl, and the other is hydrogen, allyl,alkanoyl, aryloxycarbonyl, unsubstituted alkyl, or the residue of anα-amino acid in which the hydroxy group of the carboxyl group isexcluded; —CO₂R⁹; —C(═O)NR¹⁰R¹¹; —S(═O)_(r)R³⁰, wherein R³⁰ issubstituted or unsubstituted lower alkyl, aryl, or heteroaryl; or —OR³¹,wherein R³¹ is hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted alkanoyl; —CH═N—R³², wherein R³² ishydroxyl, lower alkoxy, amino, guanidino, ureido, imidazolylamino,carbamoylamino, or NR^(26A)R^(27A) (wherein R^(26A) is the same as R²⁶and R^(27A) is the same as R²⁷); or —CH₂Q wherein Q is a sugar residuerepresented by

 wherein V represents hydrogen, methyl, ethyl, benzyl, acetyl, ortrifluoroacetyl; Y is hydrogen; —OH; —OC(═O)R³³, wherein R³³ is alkyl,aryl, or amino; —OCH₂O—alkyl; —O—alkyl; aralkyloxy; or X and Y arecombined as —X—Y to form —CH₂OCO₂— or —CH₂N(R^(16B))CO₂— (whereinR^(16B) is the same as R¹⁶); A¹ and A² are hydrogen, or both arecombined to represent O; or B¹ and B² are hydrogen, or both are combinedto represent O; or a pharmaceutically acceptable salt thereof; with theproviso that at least one of A¹,A² or B¹,B² represents O; and with thefurther proviso that both X and Y are not simultaneously hydrogen, andwherein said neurological disorder is selected from the group consistingof Alzheimer's disease, motor neuron disorders, Parkinson's disease,cerebrovascular disorders, Huntington's disease, AIDS dementia,epilepsy, multiple sclerosis, peripheral neuropathies, disorders inducedby excitatory amino acids, and disorders associated with concussive orpenetrating injuries of the brain or spinal cord.
 2. The method of claim1, wherein X is —C(═O)NR²⁶R²⁷, carboxyl, lower alkoxycarbonyl, formyl,lower alkyl, —CH₂OR³¹, —CH₂NR²⁸R²⁹, or —CH₂S(O)_(r)R³⁰.
 3. The method ofclaim 1, wherein R¹ and R² are H.
 4. The method of claim 1, wherein R³is hydrogen or a protecting group.
 5. The method of claim 1, wherein thecompound is selected from the group consisting of Compounds VI, VII,VIII, X, XII, XIV, XV, XVI, XVII, XVIII, XIX, XXV, and XXVII:


6. The method of claim 1 wherein said motor neuron disorder isamyotrophic lateral sclerosis.
 7. The method of claim 1 wherein saidcerebrovascular disorder is selected from the group consisting of strokeand ischaemia.
 8. A method of treating cancer comprising administeringto a patient an effective amount of a compound of the following formula:

wherein: R¹ and R² independently are: hydrogen; lower alkyl; halogen;acyl; nitro; sulfonic acid; —CH═NR⁴, wherein R⁴ is guanidino,heterocyclic, or —NR⁵R⁶, wherein one of R⁵ and R⁶ is hydrogen or loweralkyl, and the other of R⁵ and R⁶ is hydrogen, lower alkyl, acyl, aryl,heterocyclic, carbamoyl or lower alkylaminocarbonyl; —NR⁵R⁶; —CH(SR⁷)₂,wherein R⁷ is lower alkyl or alkylene; —(CH₂)_(j)R⁸, wherein j is 1-6,and R⁸ is halogen, substituted aryl, unsubstituted aryl, substitutedheteroaryl, unsubstituted heteroaryl or N₃; —CO₂R⁹, wherein R⁹ ishydrogen, substituted lower alkyl, unsubstituted lower alkyl,substituted aryl, unsubstituted aryl, substituted heteroaryl, orunsubstituted heteroaryl; —C(═O)NR¹⁰R¹¹, wherein R¹⁰ and R¹¹independently are hydrogen, substituted lower alkyl, unsubstituted loweralkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted aralkyl, unsubstituted aralkyl,lower alkylaminocarbonyl, or lower alkoxycarbonyl, or R¹⁰ and R¹¹ arecombined with a nitrogen atom to form a heterocyclic group; —OR¹²,wherein R¹² is hydrogen, substituted lower alkyl, unsubstituted loweralkyl, substituted aryl, unsubstituted aryl or —C(═O)R¹³, wherein R¹³ ishydrogen, NR¹⁰R¹¹, substituted lower alkyl, unsubstituted lower alkyl,substituted aryl, unsubstituted aryl, substituted heteroaryl,unsubstituted heteroaryl, substituted aralkyl, or unsubstituted aralkyl;—NR¹⁰R¹¹; —C(═O)R¹⁴, wherein R¹⁴ is hydrogen, lower alkyl, substitutedaryl, unsubstituted aryl, substituted heteroaryl, or unsubstitutedheteroaryl; —S(═O)_(r)R¹⁵, wherein r is 0 to 2, and R¹⁵ is hydrogen,substituted lower alkyl, unsubstituted lower alkyl, substituted aryl,unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,substituted aralkyl, unsubstituted aralkyl, thiazolinyl,—(CH₂)_(a)C(═O)NR¹⁰R¹¹ or (CH₂)_(a)CO₂R¹⁶, wherein a is 1 or 2, and R¹⁶is hydrogen or lower alkyl; —OR¹⁷, wherein R¹⁷ is hydrogen, lower alkyl,or —C(═O)R¹⁸, wherein R¹⁸ is substituted lower alkyl, unsubstitutedlower alkyl, substituted aryl, or unsubstituted aryl;—C(═O)(CH₂)_(j)R¹⁹, wherein R¹⁹ is hydrogen, halogen, NR¹⁰R¹¹, N₃, SR¹⁵,or OR²⁰, wherein R²⁰ is hydrogen, substituted lower alkyl, unsubstitutedlower alkyl, or C(═O)R¹⁴; —CH(OH)(CH₂)_(j)R¹⁹;—(CH₂)_(d)CHR²¹CO₂R^(16A), wherein d is 0-5, and R²¹ is hydrogen,CONR¹⁰R¹¹, or CO₂R^(16A), wherein R^(16A) is the same as R¹⁶;—(CH₂)_(d)CHR²¹CONR¹⁰R¹¹; —CH═CH(CH₂)_(m)R²², wherein m is 0-4, and R²²is hydrogen, lower alkyl, CO₂R⁹, substituted aryl, unsubstituted aryl,substituted heteroaryl, unsubstituted heteroaryl, OR¹², or NR¹⁰R¹¹;—CH═C(CO₂R^(16A))₂; —C≡C(CH₂)_(m)R²²; —SO₂NR²³R²⁴, wherein R²³ and R²⁴independently are hydrogen, lower alkyl, or groups that form aheterocycle with the nitrogen atoms; —OCO₂R^(13A), wherein R^(13A) isthe same as R¹³; or —OC(═O)NR¹⁰R¹¹; R³ is hydrogen; lower alkyl;carbamoyl; amino; tetrahydropyranyl; hydroxyl; C(═O)H; aralkyl; loweralkanoyl; or CH₂CH₂R²⁵, wherein R²⁵ is halogen, amino, di-loweralkylamino, hydroxyl, or hydroxysubstituted lower alkylamino; X ishydrogen; formyl; carboxyl; lower alkoxycarbonyl; loweralkylhydrazinocarbonyl; —CN; lower alkyl; —C(═O)NR²⁶R²⁷, wherein R²⁶ andR²⁷ independently are hydrogen, unsubstituted lower alkyl, orunsubstituted aryl; or R²⁶ and R²⁷ are combined with a nitrogen atom toform a heterocyclic group; —CH(R³⁴)W, wherein R³⁴ is hydrogen or loweralkyl, and W is —N═CHN(alkyl)₂; guanidino; N₃; NR²⁸R²⁹, wherein R²⁸ orR²⁹ is hydrogen or lower alkyl, and the other is hydrogen, allyl,alkanoyl, aryloxycarbonyl, unsubstituted alkyl, or the residue of anα-amino acid in which the hydroxy group of the carboxyl group isexcluded; —CO₂R⁹; —C(═O)NR¹⁰R¹¹; —S(═O)_(r)R³⁰, wherein R³⁰ issubstituted or unsubstituted lower alkyl, aryl, or heteroaryl; or —OR³¹,wherein R³¹ is hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted alkanoyl; —CH═N—R³², wherein R³² ishydroxyl, lower alkoxy, amino, guanidino, ureido, imidazolylamino,carbamoylamino, or NR^(26A)R^(27A) (wherein R^(26A) is the same as R²⁶and R^(27A) is the same as R²⁷); or —CH₂Q wherein Q is a sugar residuerepresented by

 wherein V represents hydrogen, methyl, ethyl, benzyl, acetyl, ortrifluoroacetyl; Y is hydrogen; —OH; —OC(═O)R³³, wherein R³³ is alkyl,aryl, or amino; —OCH₂O-alkyl; —O—alkyl; aralkyloxy; or X and Y arecombined as —X—Y to form —CH₂OCO₂— or —CH₂N(R^(16B))CO₂— (whereinR^(16B) is the same as R¹⁶); A¹ and A² are hydrogen, or both arecombined to represent O; or B¹ and B² are hydrogen, or both are combinedto represent O; or a pharmaceutically acceptable salt thereof; with theproviso that at least one of A¹,A² or B¹,B² represents O; and with thefurther proviso that both X and Y are not simultaneously hydrogen. 9.The method of claim 8, wherein X is —C(═O)NR²⁶R²⁷, carboxyl, loweralkoxycarbonyl, formyl, lower alkyl, —CH₂OR³¹, —CH₂NR²⁸R²⁹, or—CH₂S(O)_(r)R³⁰.
 10. The method of claim 8, wherein R¹ and R² are H. 11.The method of claim 8, wherein R³ is hydrogen or a protecting group. 12.The method of claim 8, wherein the compound is selected from the groupconsisting of Compounds VI, VII, VIII, X, XII, XIV, XV, XVI, XVII,XVIII, XIX, XXV, and XXVII: